Title

Author

Date of Award

2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

The eukaryotic initiation factor 3e is a subunit of the eIF3 complex that was found to mediate the interaction between eIF4G and eIF3 complex. eIF3e was initially found as one of the common integration sites of the mouse mammary tumor virus (MMTV), an integration that lead to the formation of mammary epithelial tumors in mice. Integration of MMTV into eIF3e induces the formation of truncated gene products and the potential formation of truncated forms of the protein. It has been shown that the ectopic expression of truncated eIF3e alone, without MMTV sequences or without disrupting endogenous eIF3e alleles, is sufficient to induce malignant transformation in mammalian cells and tumor formation in transgenic mice, but the mechanisms used by truncated eIF3e to cause transformation are not understood. In the present work, we investigate if 3e5 affects protein synthesis in a way that may explain its ability to transform cells and cause tumors. Two different mouse cell lines were used during the present work: HC11 and NIH3T3. In both cases, the cells were transfected with vectors that allow the constitutively expression of truncated eIF3e. The NIH3T3 model system closely mimics the integration of MMTV into only one allele of the eIF3e gene. Cells expressing truncated eIF3e exhibited reduced rates of protein synthesis, which was observed by incorporation of radio-labeled methionine. We tested the capacity of these cells to induce translation in a cap-independent manner using a bicistronic mRNA from which firefly luciferase is expressed in a cap-dependent manner whereas Renilla luciferase is expressed in a cap-independent manner from the hepatitis C virus internal ribosome entry sequence (IRES) element. Using this strategy, we observed enhanced translation of cap-independent Renilla luciferase in cells expressing truncated eIF3e. Since exogenously introduced mRNAs do not undergo a "nuclear experience" and may not behave exactly the same as endogenous mRNAs, so we also tested the polysome distribution of endogenous mRNAs. An increase in polysome size indicates an increase in initiation rate relative to elongation/termination. We found that GAPDH, actin, L32 and L34 mRNAs (cap-dependent) shifted to lower polysomes in cells expressing truncated eIF3e, while the IRES-containing mRNAs for XIAP, c-Myc, CYR61, and Pim-1 shifted to heavy polysomes. Our results support a model where expression of truncated eIF3e tips the balance toward cap-independent translation, thereby causing upregulation of known oncoproteins such as c-Myc and inhibitors of apoptosis such as XIAP..